title: Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings.
abstract: An Agro-mediated transformation method has been adapted in Catharanthus roseus seedlings for transient overexpression. Our results suggest that Agro-mediated methods may induce defense-related genes, which should be considered in its application. The Fast Agro-mediated Seedling Transformation (FAST) method, which involves the co-cultivation and transient transformation of young seedlings with Agrobacterium, was adapted and optimized in Catharanthus roseus. We investigated the optimal conditions for Gus expression by varying the Agrobacterium density (OD600 = 0.29 and 0.50), A. rhizogenes strain (15834 and R1000), and co-cultivation time in liquid (2, 12, or 24 h) followed by incubation time on solid media (1 or 2 days). Transformation efficiency was assessed quantitatively in terms of average GUS intensity per cotyledon surface area and percentage of cotyledons transformed. GUS staining was observed in 100% of cotyledons co-cultivated with A. rhizogenes (OD600 = 0.50) co-transformed with the Mas promoter-driven Gus and pSoup helper plasmids, in the presence of 0.01% v/v Silwet L-77 for 24 h in liquid followed by 2-days on solid media. In addition, we observed that co-cultivation with Agrobacterium strongly induced Zct1 and Orca3, two transcription factors known to regulate defense-related alkaloid biosynthesis in C. roseus. Homologous transcription factors regulate defense responses in many plant species. Therefore, possible induction of defense-related genes by Agro-mediated transformation should be a consideration in experimental design.
authors.affiliation: Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Weaver
authors.forename: Sheba
authors.initials: S
authors.lastname: Goklany
authors.forename: Noreen
authors.initials: N
authors.lastname: Rizvi
authors.forename: Erin J
authors.initials: EJ
authors.lastname: Cram
authors.forename: Carolyn W T
authors.initials: CW
authors.lastname: Lee-Parsons
mesh_terms: D060054:Agrobacterium / Q000502:physiology*; D030008:Catharanthus / Q000235:genetics* / Q000382:microbiology*; D018506:Gene Expression Regulation, Plant; D005821:Genetic Techniques; D005966:Glucuronidase / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D036226:Seedlings / Q000235:genetics* / Q000382:microbiology*; D014170:Transformation, Genetic
chemical_list: D010940:Plant Proteins; D005966:Glucuronidase